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. 2007;2(5):1145-51.
doi: 10.1038/nprot.2007.172.

Tandem affinity purification of functional TAP-tagged proteins from human cells

Juraj Gregan  1 , Christian G Riedel, Mark Petronczki, Lubos Cipak, Cornelia Rumpf, Ina Poser, Frank Buchholz, Karl Mechtler, Kim Nasmyth
Affiliations

Tandem affinity purification of functional TAP-tagged proteins from human cells

Juraj Gregan et al. Nat Protoc. 2007.

Abstract

Tandem affinity purification (TAP) is a generic two-step affinity purification protocol for isolation of TAP-tagged proteins together with associated proteins. We used bacterial artificial chromosome to heterologously express TAP-tagged murine Sgo1 protein in human HeLa cells. This allowed us to test the functionality of the Sgo1-TAP protein by RNA interference-mediated depletion of the endogenous human Sgo1. Here, we present an optimized protocol for purification of TAP-tagged Sgo1 protein as well as KIAA1387 from HeLa cells with detailed instructions. The purification protocol can be completed in 1 day and it should be applicable to other proteins.

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Figures

Figure 1
Figure 1
Rescue experiment. Wild-type HeLa cells (wt) and BAC-transgenic HeLa cells expressing either murine SGO1 (mSGO1) or murine SGO1-TAP (mSGO1-TAP) were transfected with two different Sgo1 siRNAs (siSgo1-A, siSgo1-B) or deionized H2O (mock). Cells were examined by chromosome spreading followed by Giemsa staining and mitotic cells were classified into two categories based on chromosome configuration.
Figure 2
Figure 2
Purified proteins visualized by silver staining. Protein complexes associated with murine Sgo1-TAP and murine KIAA1386-TAP were isolated by tandem affinity purification, separated by SDS-PAGE and visualized by silver staining. Molecular weight marker (MW marker) is indicated on the left.
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